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Summary Homozygous deletions of p16/ CDKN2A are prevalent in cancer, and these mutations commonly involve co-deletion of adjacent genes, including methylthioadenosine phosphorylase ( MTAP ). Here, we used shRNA screening and identified the metabolic enzyme, methionine adenosyltransferase II alpha (MAT2A), and the arginine methyltransferase, PRMT5, as vulnerable enzymes in cells with MTAP deletion. Metabolomic and biochemical studies revealed a mechanistic basis for this synthetic lethality. The MTAP substrate methylthioadenosine (MTA) accumulates upon MTAP loss. Biochemical profiling of a methyltransferase enzyme panel revealed that MTA is a potent and selective inhibitor of PRMT5. MTAP -deleted cells have reduced PRMT5 methylation activity and increased sensitivity to PRMT5 depletion. MAT2A produces the PRMT5 substrate S-adenosylmethionine (SAM), and MAT2A depletion reduces growth and PRMT5 methylation activity selectively in MTAP -deleted cells. Furthermore, this vulnerability extends to PRMT5 co-complex proteins such as RIOK1. Thus, the unique biochemical features of PRMT5 create an axis of targets vulnerable in CDKN2A/MTAP -deleted cancers. [ABSTRACT FROM AUTHOR]

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine locus. These mutants recapitulate essentially Aldh2 all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the allele--alcohol interaction may be an even greater ALDH2*2 human public health hazard than previously appreciated. [ABSTRACT FROM AUTHOR]

This article describes the first step toward full (that includes conditions for both absence and presence of metabolic activation) validation and drug discovery application of a 96-well, automated, high-content micronucleus (HCMN) assay. The current validation tests were performed using Chinese hamster ovary cells, in the absence of metabolic activation, against three distinct sets of drug-like compounds that represent all stages of a drug discovery pipeline. A compound categorization scheme was created based on quantitative relationships between micronucleus (MN) signals, cytotoxicity, and compound solubility. Results from initial validation compounds (n = 38) set the stage for differentiating overall positive and negative MN inducers. To delve deeper into the compound categorization process, a more extensive validation set, consisting of a larger set (n = 370) of “drug-like but less optimized” early-stage compounds, was used for further refinement of positive and negative compound categories. The predictivity and applicability of the assay for clinical stage compounds was ascertained using (n = 168) clinically developed marketed drugs or well-studied compounds. Upon full validation, a detailed analysis of results established five compound categories—NEG (negative), NEG/xx μM (negative up to the solubility limit of xx μM), WPOS (weak positive), POS (positive), and INCON (inconclusive). Furthermore, examples of lead-finding applications and ongoing investigative HCMN activities are described. A proposal is offered on how the HCMN assay can be positioned in parallel to the overall stage gates (e.g., scaffold selection, lead optimization, late-stage preclinical development) of drug discovery programs. Because of its greater throughput, 1-week turnaround time, and a substantially reduced (1–2 mg) requirement for compound consumption, the HCMN assay is appropriate for developing structure-genotoxicity relationships and for mechanistic genotoxicity studies. The assay does not replace the Organization for Economic Cooperation and Development–compliant, non-good laboratory practice in vitro MN test (e.g., slide-based MN test in TK6 lymphoblastoid cells) that is used for full characterization of lead candidates. [ABSTRACT FROM AUTHOR]

OBJECTIVE--The inhibition of gut and renal sodium-glucose cotransporters (SGLTs) has been proposed as a novel therapeutic approach to the treatment of diabetes. We have identified dapagliflozin as a potent and selective inhibitor of the renal sodium-glucose cotransporter SGLT2 in vitro and characterized its in vitro and in vivo pharmacology. RESEARCH DESIGN AND METHODS--Cell-based assays measuring glucose analog uptake were used to assess dapagliflozin's ability to inhibit sodium-dependent and facilitative glucose transport activity. Acute and multi-dose studies in normal and diabetic rats were performed to assess the ability of dapagliflozin to improve fed and fasting plasma glucose levels. A hyperinsulinemic-euglycemic clamp study was performed to assess the ability of dapagliflozin to improve glucose utilization after multi-dose treatment. RESULTS--Dapagliflozin potently and selectively inhibited human SGLT2 versus human SGLT1, the major cotransporter of glucose in the gut, and did not significantly inhibit facilitative glucose transport in human adipocytes. In vivo, dapagliflozin acutely induced renal glucose excretion in normal and diabetic rats, improved glucose tolerance in normal rats, and reduced hyperglycemia in Zucker diabetic fatty (ZDF) rats after single oral doses ranging from 0.1 to 1.0 mg/kg. Once-daily dapagliflozin treatment over 2 weeks significantly lowered fasting and fed glucose levels at doses ranging from 0.1 to 1.0 mg/kg and resulted in a significant increase in glucose utilization rate accompanied by a significant reduction in glucose production. CONCLUSIONS--These data suggest that dapagliflozin has the potential to be an efficacious treatment for type 2 diabetes. Diabetes 57:1723-1729, 2008 [ABSTRACT FROM AUTHOR]

Squalene epoxidase (SQLE), also known as squalene monooxygenase, catalyzes the stereospecific conversion of squalene to 2,3(S)-oxidosqualene, a key step in cholesterol biosynthesis. SQLE inhibition is targeted for the treatment of hypercholesteremia, cancer, and fungal infections. However, lack of structure-function understanding has hindered further progression of its inhibitors. We have determined the first three-dimensional high-resolution crystal structures of human SQLE catalytic domain with small molecule inhibitors (2.3 Å and 2.5 Å). Comparison with its unliganded state (3.0 Å) reveals conformational rearrangements upon inhibitor binding, thus allowing deeper interpretation of known structure-activity relationships. We use the human SQLE structure to further understand the specificity of terbinafine, an approved agent targeting fungal SQLE, and to provide the structural insights into terbinafine-resistant mutants encountered in the clinic. Collectively, these findings elucidate the structural basis for the specificity of the epoxidation reaction catalyzed by SQLE and enable further rational development of next-generation inhibitors. Squalene epoxidase (SQLE) is a key enzyme in cholesterol biosynthesis and is a target for hypercholesteremia and cancer drug development. Here the authors present the crystal structures of the human SQLE catalytic domain alone and bound with small molecule inhibitors, which will facilitate the development of next-generation SQLE inhibitors. [ABSTRACT FROM AUTHOR]

The inhibition of sodium-glucose transporters (SGLT) has been proposed as a novel therapeutic approach to treat diabetes. Relatively nonselective sodium-glucose cotransporter (SGLT) inhibitors such as phlorizin have been demonstrated to improve ambient plasma glucose levels as well as insulin sensitivity in diabetic animal models. Dapagliflozin (BMS-512148), a novel SGLT2 inhibitor under clinical investigation, selectively inhibits human SGLT2 vs. human SGLT1 in cell based assays (SGLT2 EC]sub 50] = 1.1 nM, SGLT1 EC[sub 50] = 1391 nM). In vivo, dapagliflozin acutely induces renal glucose excretion in normal and diabetic rats at doses of 0.1-1.0 mg/kg without hypoglycemia, improves glucose tolerance in normal rats at 1 and 10 mg/kg, and effectively lowers hyperglycemia in Zucker diabetic fatty (ZDF) rats following single oral doses ranging from 0.1-1.0 mg/kg. At a 1 mg/kg dose, the peak concentration of dapagliflozin in ZDF rat plasma was 1.2 µM with an area under the curve (AUC) over 6 hr of 5.2 µM*hr. In a 15 day study with 17 week old male ZDF rats, dapagliflozin treatment resulted in significantly lowered fasting and fed glucose levels at doses ranging from 0.1-1.0 mg/kg. Dapagliflozin treatment also improved key aspects of metabolic dysregulation in this model as evaluated in a hyperinsulinemic euglycemic clamp procedure. A significant increase in glucose infusion rate (GIR) was accompanied by a significant reduction in hepatic glucose production (HGP) when measured on the third day following the final dose. These data suggest that dapagliflozin has the potential to be an efficacious treatment for human type 2 diabetes. [ABSTRACT FROM AUTHOR]